Bottom Line:
Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA.These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate.Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

ABSTRACTThe regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

Figure 6: Steady state levels of proteins involved in mitochondrial clearance and biogenesis following ethidium bromide exposure. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, total cellular protein extracts were prepared and analyzed by immunoblot as described in Section “Materials and Methods” for a subset of mitochondrial resident proteins. The levels of beta-actin or beta tubulin are presented as a control for equal protein loading. The immunoblot presented is a representative blot of a minimum of two measurements with similar results.

Mentions:
We examined proteins associated with autophagy in HDF cultures exposed to ethidium bromide. The steady state level of p62 sequestasome 1 (p62/SQSMTM1) increased in HDF cultures exposed to ethidium bromide but was uniformly lower in HDF cultures that had been treated with rapamycin (Figure 6). Both the unconjugated form of microtubule-associated protein light chain 3(LC3B-I) and the conjugated form of LC3 (LC3B-II) decreased following exposure to ethidium bromide (Figure 6), while rapamycin treated cultures showed reduced levels of both LC3B-I and LC3B-II that were not affected by exposure to ethidium bromide. We also examined the steady state levels of Parkin, a protein involved in mitochondrial turnover (30–32). The steady state levels of Parkin increased following exposure to ethidium bromide, while Parkin levels were uniformly elevated in rapamycin treated cultures relative to control cultures (Figure 6). The levels of both NRF1 and NFE2L2 (NRF2) were increased following exposure to ethidium bromide. Rapamycin treatment increased the levels of both proteins in normal growth conditions, consistent with our previous observations (29), while ethidium bromide exposure did not increase the levels of NRF1 and NFE2L2 further (Figure 6).

Figure 6: Steady state levels of proteins involved in mitochondrial clearance and biogenesis following ethidium bromide exposure. Human fibroblast cell cultures grown in the presence or absence of 1 nM rapamycin were exposed to increasing doses of ethidium bromide for a period of 7 days. At this time, total cellular protein extracts were prepared and analyzed by immunoblot as described in Section “Materials and Methods” for a subset of mitochondrial resident proteins. The levels of beta-actin or beta tubulin are presented as a control for equal protein loading. The immunoblot presented is a representative blot of a minimum of two measurements with similar results.

Mentions:
We examined proteins associated with autophagy in HDF cultures exposed to ethidium bromide. The steady state level of p62 sequestasome 1 (p62/SQSMTM1) increased in HDF cultures exposed to ethidium bromide but was uniformly lower in HDF cultures that had been treated with rapamycin (Figure 6). Both the unconjugated form of microtubule-associated protein light chain 3(LC3B-I) and the conjugated form of LC3 (LC3B-II) decreased following exposure to ethidium bromide (Figure 6), while rapamycin treated cultures showed reduced levels of both LC3B-I and LC3B-II that were not affected by exposure to ethidium bromide. We also examined the steady state levels of Parkin, a protein involved in mitochondrial turnover (30–32). The steady state levels of Parkin increased following exposure to ethidium bromide, while Parkin levels were uniformly elevated in rapamycin treated cultures relative to control cultures (Figure 6). The levels of both NRF1 and NFE2L2 (NRF2) were increased following exposure to ethidium bromide. Rapamycin treatment increased the levels of both proteins in normal growth conditions, consistent with our previous observations (29), while ethidium bromide exposure did not increase the levels of NRF1 and NFE2L2 further (Figure 6).

Bottom Line:
Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA.These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate.Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

ABSTRACTThe regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.